“Serpin” is a name given to members of a group of single-chain 40-60 kDa proteins many of which are serine protease inhibitors, an activity from which the family originally derived its name (for reviews, see for example, Bird, Results Probl Cell Differ 24:63-89 (1998); Pemberton, Cancer J 10(1):1-11 (1997); Worrall et al., Biochem Soc Trans 27(4):746-50 (1999); and Irving et al., Genome Res 10:1845-64 (2000)). Serpins are conserved at the primary amino acid sequence level and also in their tertiary structure. Serpin family members generally share about 15-50% amino acid sequence identity. Three-dimensional computer generated models of the serpins are virtually superimposable. Serpins are found in vertebrates and animal viruses, plants and insects, and identified members of this superfamily number nearly 300.
Serpins may localize to the intracellular or extracellular space, the latter being mediated by a classical N-terminal signal sequence. A subset of the serpin family, the ovalbumin-like serpins (or “ov-serpins”), have a non-cleavable facultative signal sequence found near the N-terminus (Remold-O'Donnell, FEBS Letters 315:105-108 (1993)). Ov-serpins that possess this non-canonical signal sequence can demonstrate dual localization inside and outside the cell and are suspected to inhibit different intracellular and extracellular proteases. An example of a serpin with dual localization is PAI-2. Regulation of this dual localization may result in elevated plasma levels associated with various pathologies, such as SCCA in squamous cell carcinoma (Pemberton, 1997).
Serine proteases, which provide the targets for many of the inhibitory serpins, are involved in, and regulate, many aspects of biology including: degradation of extracellular matrix (such as elastases), vascular hemostasis (such as thrombin in coagulation, plasmin in thrombolysis), complement activation (such as complement factors), vasodilation in inflammation and hypertension (such as kallikreins), and digestion (such as trypsin). Leukocytes produce and store in vesicles many different serine proteases involved in cytotoxic responses (e.g. granzymes, chymases). Serpins also play a role in cell migration.
Serpin family members participate in variety of intracellular and extracellular processes, including serving as chaperones for protein folding, storage proteins, and transporting hormones. Inhibitory serpins participate in many important biological activities, including: complement activation; fibrinolysis; coagulation; cellular differentiation; tumor suppression; and selection processes associated with tumor survival (i.e., apoptosis and cell migration). Mutations in serpins may cause a number of diseases, some of which are associated with serpin polymerization (Irving et al., 2000). Such diseases include, for example, blood clotting disorders, emphysema, cirrhosis and dementia.
Many serpins are found at relatively high levels in human plasma. Plasma serpins are variably glycosylated, though this glycosylation may not be required for activity (Potempa et al., J Biol Chem 269:15957 (1994)). These include α1 antitrypsin (α1AT), which is involved in restructuring of connective tissue; C1 inhibitor, which controls complement activation; plasminogen activator inhibitors 1 and 2 (PAI-1 and PAI-2), which help control fibrinolysis; and antithrombin, which is involved in regulating the coagulation cascade. Also present in blood are angiotensinogen, which when cleaved gives rise to vasopressor peptide that helps control blood pressure, as well as thyroxine binding globulin (TBG) and the corticosteroid binding globulin (CBG). Proteolytic cleavage of TBG appears to provide a mechanism for site-specific release of thyroxine (Schussler, Thyroid 10(2):141-49 (2000)). The serpins maspin, PAI-2 and (α1AT, under certain circumstances are capable of polymerizing (Pemberton, 1997). Some serpins, such as AT-III, achieve a much higher level of inhibitory activity if activated by polysulfated oligosaccharides such as heparin (Potempa et al., 1994). Other serpins shown to bind heparin cofactor II include protease nexin-1, active protein C inhibitor and PAI-1 (Potempa et al., 1994).
The ov-serpins are characterized by their relatively high degree of homology with chicken ovalbumin. The ov-serpins are reviewed, for example, in Worrall et al., 1999 and in Remold-O'Donnell, 1993. Ov-serpins generally have eight exons, seven introns and highly conserved intron-exon boundaries, though the ov-serpin PI-6 has only seven exons and six introns. The ov-serpins typically lack the extended N-terminal and C-terminal regions found in other serpins. Moreover, they possess an internal hydrophobic sequence near the amino terminus that allows both secretion and intracellular retention, depending on the cell type or the state of differentiation of the cell in which the protein is expressed. Ov-serpins have a higher degree of amino acid homology with one another than with the other serpins (e.g., they are 40% to 50% homologous with each other, but only about 30% homologous with the other serpins). In addition, ov-serpins have a penultimate serine at the C-terminus, and they have nearly identical splice-junction positions. The ov-serpins are predominantly intracellular, though some are secreted as well as being found intracellularly (e.g., maspin and PAI-2).
Other physiological processes in which serpins have been implicated include prevention of tumor invasiveness (maspin), storage (ovalbumin) and functioning as a chaperone in protein folding (HSP47) (see, for example, Whisstock et al., Trends Biochem Sci 23(2):63-67 (1998); Sauk et al., Connective Tissue Res 37 (1-2): 105-119 (1998)). The heat shock protein HSP47, although studied primarily for its role in collagen processing, sometimes escapes from the endoplasmic reticulum and reaches the cell surface, thus prompting Sauk et al. to propose that it could modulate cell migration during development and/or metastatic invasion of cancer cells (Sauk et al., 1998).
The clinical manifestations of serpin dysfunction include emphysema and cirrhosis (Whisstock et al., 1998; Bird, 1998), which are associated with deficiencies in α1-proteinase inhibitor (also called “α1-antitrypsin”), which ordinarily control alveolar damage by neutrophil elastase. Accumulation of α1-proteinase inhibitor mutants in liver can give rise to hepatitis or cirrhosis (Bird, 1998). Defective antithrombin III may underlie recurrent thromboembolic disease, and certain bleeding disorders could be related to deficient α2-antiplasmin activity, which results in higher levels of active plasmin thus increased fibrinolysis, while other clinical manifestations of serpin dysfunction include thrombosis, associated with antithrombin, which targets thrombin thereby inhibiting the coagulation cascade (Bird, 1998). It has been noted also that mutations in antithrombin III and (α2-antiplasmin are associated with uncontrolled coagulopathies, and that hereditary angioneurotic edema is associated with deficiencies in C1-inhibitor, which targets C1-elastase and is an enzyme involved in the complement cascade (Potempa et al., 1998; Whisstock, 1998).
It has been noted that many aspects of osteoarthritis and rheumatoid arthritis involve cell invasion, that is, the ability of cells to cross anatomical barriers separating tissue compartments, and that proteases such as plasminogen activators and the matrix metalloproteinases play a role in controlling the activity of invading and proliferating cells in inflamed joints (Del Rosso et al., Clin Exp Rheumatol 17:485-98 (1999)). Del Rosso et al. summarize evidence that urokinase plasminogen activator (uPA) plays a key role in extracellular matrix destruction and formation of lesions in arthritic joints. They suggest that pharmacologically controlling the plasminogen activating system may be a viable approach to preventing the development of bone lesions and joint ankylosis in arthritis.
The serpin family also includes viral proteins that play a role in viral virulence. For example the cowpox cytokine response modifier gene (CrmA) can block apoptosis induced by a variety of stimuli, and is known to inhibit several of the interleukin-1β converting enzymes (ICE-like cysteine proteases). CrmA is considered a virulence factor for the cowpox virus. SERP1 (myxoma virus) targets uPA, tissue plasminogen activator (tPA) and plasmin, and promotes myxoma virus virulence.
The ov-serpins appear to be clustered within a 500 kb region telomeric to BCL2 at 18q21.3 (Silverman et al., Tumor Biol 19:480-87 (1998)). The two SCCA genes are less than 10 kb apart in this region and are flanked by the genes encoding PAI-2 and maspin (also called SERPINB5 or PI5). Additional serpins mapping to 18q21.3 are the cytoplasmic antiproteinase 2 (CAP2, also called PI8), bone marrow-associated serpin (bomapin, also called PI10 or serpin B10), hurpin (also called SERPINB13 OR “headpin”) and megsin. The order of several of these serpins is cen-maspin, hurpin, SCCA-2, SCCA-1, megsin, PAI-2, bomapin and CAP2-tel. The SCCA-2 coding region has been cloned, and is disclosed in WO 9714425. Contigs containing this gene cluster can be found at the NCBI website using the nucleotide search and entering one of the following contig numbers: AC019355; AP001404; or AC015536. Chromosome 18q is known to be associated with breakpoints and loss of heterozygosity in cancers of the head and neck and other malignancies, thus suggesting that intact functioning of the serpin genes within this cluster may be disadvantageous to tumor growth (Spring et al., Biochem Biophys Res Comm 264:299-304 (1999)).
Some of the serpins have no discernable protease inhibitory activity, while others have been shown to inhibit serine or cysteine proteases (see, for example, Pemberton, 1997). Most of the ov-serpins inhibit serine proteases, however, SCCA-1, for example, inhibits cysteine proteases such as papain, cathepsins L, S and K, while the closely related SCCA-2 (92% amino acid sequence identity) inhibits chymotrypsin-like serine proteases such as mast cell chymase and cathepsin G. SCCA-1 is found mainly inside of cells, while the more acidic SCCA-2 is largely expressed in squamous cell carcinoma and released outside the cells (Suminami et al., Tumor Biol 19:488-93 (1998)). The cowpox CrmA protein also is a cysteine proteinase inhibitor. Hurpin is predicted to be an inhibitory serpin based on its hinge region homology with other serpins that possess this type of activity (Spring et al., (1999).
The basic scaffold possessed by all serpins usually includes nine α helices and three β-pleated sheets. Serpins that inhibit proteinases do so via a reactive site loop or “RSL” of about 20 to 30 amino acids located 30 to 40 amino acids from the carboxy terminus. The RSL is exposed on the surface of the protein and is susceptible to cleavage by non-target proteases (see, for example, Potempa et al., 1994). The core structure of the serpin molecule folds into a three-β-sheet pear shape that presents the RSL at the top of the structure. The RSL contains “bait” sequences that are believed to mimic the target proteinase's substrate. The inhibitory serpins regulate the activity of specific serine proteases by mimicking the protease's substrate and covalently binding to the protease when cleaved at the RSL. Upon cleavage by the target protease, inhibitory serpins undergo a dramatic conformational change, called the “stressed-to-relaxed” transition, which is accompanied by the insertion of the remaining reactive site loop into one of the β sheets. During this transition, serpins form a stable heat-resistant complex with the target protease. The sequence of the RSL, and particular the P1 and adjacent amino acid residues, determine an inhibitory serpin's specificity for a protease. An RSL is considered a key feature of serpin family members, and this structure is presented in the exposed surface loop at the top of the protein even in serpins that are not known to inhibit any proteinases.
Serpins with inhibitory activity possess several regions important in controlling and modulating serpin conformational changes associated with attaching to a target protease. As summarized in Irving et al. (2000), these are the hinge region (the P15-P9 portion of the RSL); the breach (located at top of the A β-sheet, the point of initial insertion of the RSL into the A β-sheet); the shutter (at top of the A β-sheet, the point of initial insertion of the RSL into the A β-sheet); and the gate (including strands s3C and s4C; to insert into the A β-sheet, the RSL must pass around the β-turn linking strands s3C and s4C). Inhibitory serpins possess a high degree of conservation at many key amino acid residues located in the above regions which that are believed to be necessary for enabling the protein to undergo the stressed to relaxed transition (see, for example, Table 2 in Irving et al., 2000).
Serpins lacking protease inhibitory function may exploit their “bait” sequences to attract a proteinase that cleaves within the bait sequence to activate a biological effector. Leukocyte elastase inhibitor (LEI), for example, appears to be converted by the serine protease elastase into a deoxyribonuclease that functions to degrade DNA during apoptosis (discussed in WO 99/58560). Another serpin, thyroxine binding globulin, is proteolytically cleaved to release biologically active T4 at specific locations in the body (Schussler, 2000) and angiotensinogen present in serum is cleaved by its target proteinase to generate the biologically active angiotensin protein. Similarly, corticosteroid binding protein is cleaved by the elastase at inflammatory sites to locally release corticol (Schussler, 2000).
The serpins PAI-1 and PAI-2 are involved in regulating the proteolytic breakdown of the extracellular matrix. Additionally, experiments have shown that PAI-2 protects cells against apoptosis induced by TNFα, apparently by blocking a protease, though PAI-2 does not protect against other apoptotic signals (for review, see Bird, 1998). PAI-2 also has been shown to bind to the anti-inflammatory and growth regulatory lipocortins (annexins). PAI-2 thus may be involved in regulating inflammation or growth factor signaling.
Proteinase inhibitor-9 (PI-9) is an ov-serpin proposed to protect cytotoxic T lymphocytes and natural killer cells from self-induced apoptosis resulting from exposure to granzyme B, an enzyme these lymphocytes produce to induce DNA degradation in target cells (Bird, 1998). PI-9 is not secreted and is apparently restricted to lymphoid tissue. Another inhibitory serpin, protease nexin I (PN-1) is secreted and is a potent heparin-dependent thrombin and urokinase inhibitor (Bird, 1998). It is proposed that PN-1 balances the action of thrombin on neuronal cells, thereby rescuing neural cells from apoptosis that otherwise would be induced by the action of thrombin on receptors on the surface of the neurons (Bird, 1998).
Serpins were originally shown to be involved in suppressing tumor invasion by directly inhibiting the matrix-degrading serine proteases uPA and plasmin produced by some tumor cells. Tumor-produced proteases are believed to facilitate a tumor's ability to metastasize, thus are targets for therapeutic intervention. Some cysteine proteases, such as the calpains, have been implicated in apoptotic pathways involved in tumor surveillance (Pemberton, 1997).
One serpin with demonstrated tumor-suppressing capacity is the ov-serpin maspin. Maspin is found mainly in the membrane fraction of epithelial cells (such as breast and prostate), and its expression is downregulated in mammary tumor epithelium (reviewed in Sager et al., in “Chemistry and Biology of Serpins,” eds. Church et al., Plenum Press, NY, 1997, at pages 77-88). Although maspin has been shown to suppress the invasiveness of both breast and prostate tumor cells, it does not appear to inhibit any proteases. Even so, if trypsin is used to cleave the maspin RSL, maspin loses its ability to inhibit tumors. Evidently, maspin interferes with tumor growth by some as-yet-unidentified mechanism that requires an intact RSL.
In some cancers, elevated plasma levels of certain serpins serve as markers of cancer progression. For example, the level of the prostate specific antigen (PSA) in complex with α1AT is used to monitor the progression of prostate cancer (Pemberton, 1997). Another serpin used as a tumor marker for prostate cancer is prostapin, which is described in WO 99/58560. The ov-serpins SCCA-1 and SCCA-2 in fact were originally identified as squamous cell carcinoma antigens, and a monoclonal antibody with which both SCCA's react is commonly used to monitor progression of this type of tumor (Barnes et al., Gynecol Oncol 78:62-66 (2000)). The SCCAs are elevated in squamous cell carcinomas of cervix, lung and esophagus, and SCCA levels are used as a serological marker for the extent of disease in advanced cases of these tumors (Silverman et al., 1998; Barnes et al., 2000). Suminami et al. (1998) report that the SCCA produced in epithelial cancers is primarily SCCA-2, and propose that SCCA-2 normally protects epithelial cells from inflammation. Elevated serum levels of SCCA have also been observed in patients with benign skin disorders with an inflammatory component. Such conditions include psoriasis and eczema (Barnes et al., 2000). SCCA-1 and SCCA-2 are elevated in psoriatic epidermis and are disclosed as psoriasis markers called “psoriastatin 1” and “psoriastatin 2” (WO 97/14425). Another related serpin, hurpin, also is overexpressed in psoriatic skin lesions and is disclosed as a lung tumor antigen (WO 99/47674). Hurpin is expressed in normal oral mucosal tissue, skin and in cultured keratinocytes, but is underexpressed in squamous cell cancers of the oral cavity (Spring et al., 1999). Bomapin is expressed specifically in the bone marrow (Riewald and Schleef, J Biol Chem 270:26754-57 (1995)).
Various serpins are expressed by many tissues in the body (see, for example, Worrall et al., 1999). Those present at high concentrations in the blood generally are synthesized in the liver. PAI-2 and LEI, for example, are expressed in monocytes. Maspin is expressed in normal mammary epithelium (Sager et al., 1997). SCCA-1 and SCCA-2 are expressed in normal and malignant squamous epithelium, particularly in the spinous and granular layers of epidermis and in the intermediate layer of the ectocervical epithelium (Suminami et al., 1998).
In order to develop more effective treatments for conditions and diseases mediated by serpins and their targets, more information is needed about unidentified members of the serpin polypeptide family.